This invention relates to an electrode for a dielectophoretic apparatus, in which a background can be reduced to enhance an S/N (Signal/Noise) ratio in detecting a substance to be measured (molecules to be measured) by a fluorescent strength or the like, a method for manufacturing the same, an electrode constitution provided with the electrode, and a method for separating substances using the electrode.
This invention further relates to an dielectrophoretic apparatus having an enhanced collecting ability, a method for manufacturing the same, and a method for separating substances using the apparatus.
Processing technology of materials at scales of nanometer to micrometer by means of micromachining technology such as photolithography has recently been established by development of semiconductor technologies and it has still continued its progress at present.
In the fields of chemistry and biochemistry, new technology called a Micro Total Analysis System (μ-TAS), Laboratory on a chip is growing, in which such micromachining technology is employed to carry out a whole series of chemical/biochemical analytical steps of extraction of component(s) to be analyzed from biological samples (extraction step), analysis of the component(s) with chemical/biochemical reaction(s) (analysis step), and subsequent separation (separation step) and detection (detection step) using a highly small analytical device integrated on a chip having each side of a few centimeters to a few ten centimeters in length.
Procedures of the μ-TAS are expected to make a large contribution to saving the analyzing time, reducing the amounts of samples to be used and reagents for chemical/biochemical reactions, and reducing the size of analytical instruments and the space for analysis in the course of all the chemical/biochemical analytical steps.
For the separation step in μ-TAS, in particular, there have been developed capillary electrophoretic methods in which a capillary (fine tube) with an inner diameter of less than 1 mm which is made of Teflon, silica, or the like as material is used as the separating column to achieve separation with charge differences of substances under a high electric field, and capillary column chromatographic methods in which a similar capillary is used to achieve separation utilizing the difference of the interaction between carrier in the column medium and substances.
However, capillary electrophoretic methods need a high voltage for separation and have a problem of a low sensitivity of detection due to a limited capillary volume in the detection area and also these is found such a problem that they are not suitable for separation of high molecular weight substances, though suitable for separation of low molecular weight substances, since the length of capillary for separation is limited on the capillary column on a chip and thus a capillary can not be made into a length enough for separating high molecular weight substances. In addition, in capillary column chromatographic methods there is a limit in making the throughput of separation processing higher and also there is such a problem that reducing the processing time is difficult.
Thus, attention has recently been paid to a method for solving the problems as described above, which comprises utilizing such a phenomenon so-called dielectrophoretic force that a positive and negative polarization occurs in substances placed under a non-uniform electric field , thereby providing a driving force of moving the substances [H. A. Pohl, “Dielectrophoresis”, Cambridge Univ. Press (1978); T. B. Jones, “Electromechanics of Particles”, Cambridge Univ. Press (1995), and the like].
These separation methods are presently believed to be the suitable separation method in (μTAS from the following points: (1) a rapid separation can be expected at a low applied voltage without requiring a high voltage as in capillary electrophoresis, since an electric field and its gradient can be increased to an extreme extent if micromachined electrodes are employed, because the degree of dielectrophoretic forces depends on the size and dielectric properties of substances (particles) and is proportional to the electric field gradient; (2) an increase in temperature due to applying the electric field can be minimized, since a strong electric field area is localized at a significantly small region, and a high electric field can be formed; (3) as the dielectrophoretic force is a force proportional to the electric field gradient, the force is understood as independent on the polarity of the applied voltage, and thus works under an AC electric field in a similar way to a D.C. electric field, and therefore if a high frequency A.C is employed, an electrode reaction (electrolytic reaction) in an aqueous solution can be suppressed, so that the electrodes themselves can be integrated in the channel (sample flow path); (4) improvement in a detection sensitivity can be expected, since there is no restriction to a chamber volume of the detection component unlike the capillary electrophoresis, and the like.
The dielectrophoresis termed herein is a phenomenon in which neutral particles move within non-uniform electric field, and the force exerting on molecules is called a dielectrophoretic force. The dielectrophoretic force is divided into two forces, i.e., a positive dielectrophoretic force in which substances move toward a high electric field, and a negative dielectrophoretic force in which substances move toward a low electric field.
(General Equation of Dielectrophoretic Forces)
The equivalent dipole moment method is a procedure of analyzing dielectrophoretic forces by substituting induced charges for an equivalent electric dipole. According to this method, the dielectrophoretic force Fd acted upon a spherical particle with a radius of a which is placed in an electric field E is given by:Fd=2πa3∈mRe[K*(ω)]∇(E2)  (1)wherein K*(ω) means by using an angular frequency of the applied voltage ω and the imaginary unit j as follows:K*(ω)=∈p*−∈m*/∈p*+2∈m*  (2) ∈p*=∈p−jσp/ω, ∈m*=∈m−jσm/ω  (3)wherein ∈p, ∈m, σp, and σm are permittivity and conductivity of the particle and the solution, and complex quantities are designated by *.
Equation (1) indicates that in a case of Re[K*(ω)]>0, the force works in such a way as attracting the particle toward a strong electric field side (positive dielectrophoretic, positive DEP), and in a case of Re[K*(ω)]<0, the force works in such a way as pushing the particle toward a weak electric field side (negative dielectrophoretic, negative DEP).
As will be apparent from the above-described Equations, whether the positive electrophoresis occurs in a certain substance or the negative electrophoresis occurs therein is decided by the interaction of three parameters, i.e., 1) frequency of an electric field applied, 2) conductivity and permittivity (dielectric constant) of medium, and 3) conductivity and permittivity (dielectric constant) of substance.
When these parameters are changed, even the same substance shows a positive dielectrophoresis or a negative dielectrophoresis. The negative dielectrophoresis is a phenomenon in which the substance moves toward a low electric field which is weak in density of electric flux line while the positive dielectrophoresis moves toward a high electric field which is high in density of electric flux line. FIG. 1 is a view for explaining the negative dielectrophoresis. The negative dielectrophoretic force is a force for carrying substances to such a field as to be lowered where the density of electric flux line received by the substance.
Sometimes, the substances are measured by concentrating them in an area where an electric field on an electrode is weak by using the negative dielectrophoresis as described and thereafter measuring them by fluorescent strength or the like The detection of the fluorescent strength is carried out by irradiating an excitation light on the substance to be measured to observe fluorescent light from the upper surface of the electrode.
At that time, where a conventional electrode is used, there poses a problem that the excitation light is reflected even on the electrode which is present under the substance to be measured, and thus reflected light is detected as a great background. This leads to a problem of reducing the measurement sensitivity. Besides, where a conventional electrode is used, since light does not permeate through the electrode, the substances concentrated (gathered) on the electrode cannot be detected by absorbance.
Further, the dielectrophoresis is contemplated to be a separation method suitable for μ-TAS. However, In consideration of a case of application of the dielectrophoresis to μ-TAS, it is extremely important to enhance the collecting ability. In this respect, the conventional dielectrophoretic apparatus should not yet be satisfied.
That is, if the collecting ability of substances is enhanced, separation becomes enabled in the electrode region, and the substances are held efficiently, whereby separation with high S/N (Signal/Noise) ratio is realized. Further, for example, particularly, in the Field-Flow fractionation for carrying out separation by the interaction of the dielectrophoretic force and the fluid drag exerting on the substances, separation in a short electrode region can be made even at the same flow velocity.